Detection of enzyme activity with DNA loops

ABSTRACT

The stiffness and topology of ultra-small circular DNAs and DNA/peptide hybrids are exploited to create a transducer of enzyme activity with low error rates. The modularity and flexibility of the concept are illustrated by demonstrating various transducers that respond to either specific restriction endonucleases or to specific proteases. In all cases the output is a DNA oligo signal that, as we show, can readily be converted directly to an optical readout, or can serve as input for further processing, for example, using DNA logic or amplification. By exploiting the DNA hairpin (or stem-loop) structure and the phenomenon of strand displacement, an enzyme signal is converted into a DNA signal, in the manner of a transducer. This is valuable because a DNA signal can be readily amplified, combined, and processed as information.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional PatentApplication No. 62/833,953 filed Apr. 4, 2019 and is a division of U.S.patent application Ser. No. 16/848,286 filed Apr. 14, 2020, now U.S.Pat. No. 11,067,508, the entirety of each of which is incorporatedherein by reference.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

The United States Government has ownership rights in this invention.Licensing inquiries may be directed to Office of Technology Transfer, USNaval Research Laboratory, Code 1004, Washington, D.C. 20375, USA;+1.202.767.7230; techtran@nrl.navy.mil, referencing NC 110,926.

BACKGROUND

Enzymes are protein catalysts vital to nearly all biology, allowingnature to perform the myriad room-temperature or near-room-temperaturebiochemical syntheses that make life possible. A measure of theimportance of these processes is that enzymes constitute one quarter ofthe translation products of the human genome (roughly 500 genes).Furthermore, enzymes are not only key elements of healthy cellactivities, but they are also crucial for disease processes and can thusserve as markers for these disease states. As a result, the ability todetect and monitor enzymes such as proteases, esterases, kinases, etc.is a crucial task in a multitude of applications in biology andmedicine.

A wide range of enzyme detection systems exist for use in applicationsin biomedicine, food, etc. Such methods, almost entirely in vitro, canbe grouped by the nature of their readouts and the two main classes haveoptical and electrical outputs. Performance and cost are the two maincriteria, and new approaches that have the potential to impact/improvein either of these areas are always welcome. In addition, ideas thathave the potential to function in vivo, and can add significantsophistication are of much interest for their potential to broadencapabilities.

A need exists for new techniques for detecting enzyme activity.

BRIEF SUMMARY

Described herein is a technique for enzyme detection/transductioninvolving conversion to a DNA signal that can in turn be combined,processed, and/or amplified using known DNA methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the concept of the nucleic acid loopsensor/transducer.

FIG. 2 shows a loop sensor design for endonuclease.

FIG. 3A is a gel demonstrating the desired behavior of the HaeIII loopsensor. FIG. 3B shows photoluminescence data demonstrating confirmingthe expected behavior of the HaeIII loop sensor while FIG. 3C shows thebehavior of the system over time.

FIGS. 4A-4C show operation of protease sensors.

FIGS. 5A-5D illustrate the operation of OR/NAND logical gates.

FIG. 6 shows DNA logic capable of implementing either OR or AND with aphotoluminescent output.

FIG. 7 illustrates a DNA amplification scheme.

DETAILED DESCRIPTION Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used herein, the singular forms “a”, “an,” and “the” do not precludeplural referents, unless the content clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

Overview

While DNA is of course the basis of genetics, it has also given rise tothe field of DNA nanotechnology [1] wherein the information content ofthe DNA (i.e., its base sequence) is used to program form and/orfunction for a variety of potential non-biological applications. Asdescribed herein, an enzyme signal is converted into a DNA signal, inthe manner of a transducer. This is valuable because a DNA signal can bereadily amplified, combined, and processed as information. In doing so,two important concepts of relevance are the DNA hairpin (or stem-loop)and strand displacement. Both arise commonly in nature, and both arewidely exploited in DNA nanotechnology, e.g., as molecular beacons [2]and for toehold-mediated strand displacement [3]. These capabilities arealso utilized jointly for purposes of DNA logic [4] and DNAamplification [5]. The invention disclosed here makes use of all ofthese ideas.

The subject invention is of this type in that the DNA is employed asboth a constructional and a computational material. From a structuralstandpoint, the transducer is composed largely of DNA. In this it takesparticular advantage of two key characteristics of DNA. One is itsstiffness that arises from the base-stacking of double-stranded (ds) DNAand that causes DNA to remain straight when shorter than about 15 nm(coherence length). The other relevant property of DNA is its helicalnature which can impose topological constraints on the ability of twostrands of DNA to hybridize.

A schematic of the loop sensor concept is shown in FIG. 1. As seen, thebasic design of the enzyme-to-DNA transducer consists of the circularDNA or DNA/peptide structure plus an output gate that in simplest formis a conventional molecular beacon. The circular assembly is termed herea ‘loop transducer’ and, as seen in the figure, comprises three distinctfunctional domains. The first is a stiffening domain wherein the blueloop strand has a complementary DNA strand hybridized to it to form aduplex on one side of the loop. Being double-stranded, this section isstiffened by the well-known stacking interactions, and because itslength is typically at least half of the total, it keeps the entire loopopen with the remaining portions stretched in a ‘bow’ configuration asshown in FIG. 1. The second domain of the loop transducer is thecleavage domain that contains the target of the enzyme. The cleavagedomain can be single- or double-stranded DNA, RNA, a peptide, orcombinations thereof. The third domain is the hybridizing domain whichis mostly or all single-stranded DNA that is complementary to DNA in theoutput gate (on the right in FIG. 1). While the cleavage domain remainsintact, the hybridization domain is prevented from hybridizing to theoutput gate by (i) the aforementioned stress in the loop and (ii) thetopological constraint inherent in intertwining two small loops. As aresult, if and when the enzyme cleaves the loop, it springs open,relieving both the loop stress and the topological constraint, and thusmakes the DNA of the hybridizing domain available to infiltrate and openthe hairpin in the output gate by strand displacement. As a result, thefluorophore and quencher pair at the ends of the output gate becomeseparated, and the fluorophore now provides a fluorescent signal.

The released hybridizing domain constitutes the DNA output of the looptransducer, and the conversion of this output to a fluorescent signal bythe molecular beacon represents the action of this embodiment of theoutput gate (more complicated output gates are described below). Withinthe concept just described there are many aspects that can be varied andthese may be regarded as of two types, those made with functionality inmind and those associated with optimization. While functionality isobviously of most interest, achieving functionality requiresoptimization: for example, if the hybridization strand is too short,then the hairpin will never or almost never be open, and vice versa toolong a hybridization sequence will tend to leave the hairpin always oralmost always open.

A loop sensor design suitable for detecting endonucleases is shown inFIG. 2. An endonuclease is a type of esterase that cleaves (hydrolyzes)a phosphodiester bond in double stranded DNA (dsDNA) at a specificlocation determined by the base sequence (with the target and degree ofspecificity dependent on the particular endonuclease). In this case, theentire structure is formed of DNA. The longest strand (denoted LL) istypically 80 nucleotides long but the length can be varied as desired(for example to within the range of 60 to 100 or even more nucleotides).The loop is closed with the addition of the stiffening strand(abbreviated as LS). This leaves a nick in the loop that, as verified bymolecular dynamics simulation and by experiment, does not compromise thestiffness of the loop transducer so long as the sequence is chosen sothat the stacking interaction at the nick is sufficient to maintain therigidity of the duplex [6]. Nevertheless, from the perspective ofsusceptibility to melting or degradation (e.g., by enzymatic attack ifused in serum or in vivo), one may desire not to have the nick, and itcan be eliminated using a T4 ligase. The molecular dynamics simulationswere also used as check that the stiffened designs were within theelastic limit of the dsDNA and that buckling was unlikely to occur.

The cleavage domain can be varied to detect different substrates. In thecase of an endonuclease sensor, the target sequence should bedouble-stranded, and this is done by the addition of the LC strand seenin FIG. 2. For protease sensors, this cleavage domain is the peptidesubstrate of the enzyme(s) of interest. Other possible targets thatcould be incorporated in the cleavage domain include mRNA or an RNAaptamer.

EXAMPLES Nuclease Sensing

A loop sensor design suitable for detecting endonucleases was made asshown in FIG. 2, with {LL}=80, {LS}=47, {LC}=10, {h_1}=8 and {h_2}=8,and {c}=15, where the bracket notation denotes strand length innucleotides The loop is closed with the addition of the stiffeningstrand (abbreviated as LS). This leaves a nick in the loop that, asverified by molecular dynamics simulation and by experiment, does notcompromise the stiffness of the loop transducer so long as the sequenceis chosen so that the stacking interaction at the nick is sufficient tomaintain the rigidity of the duplex. If desired, the nick can beeliminated using a ligase such as T4 ligase.

Experiments were conducted to evaluate the optimal lengths of sequencesforming the transducer and output gate.

The first aspect of the design to be considered for optimization was thehybridizing domain, in particular varying {h₁} in the range from 8 to 15and {h₂} in the range from 12 to 27. For the experiments the targetsequence in the cleavage domain was that appropriate for HaeIII (GGCC)with the loop design kept fixed with {LL}=80, {LS}=40, and {LC}=20 andonly the output gate varied (and simply shifting where the fixedsequences a₂+h₁ and b₁+h₂ divide as {h₁} and {h₂} change). Using gelelectrophoresis, the various designs were assessed in the presence orabsence of the target enzyme. Remarkably, all of the hybridizing domaindesigns worked well, in all cases showing low levels of both falsepositives (in the absence of enzyme) and false negatives (in thepresence of enzyme). This means that there is considerable flexibilityin the design of the output gate and of the hybridizing domain.Moreover, it demonstrates the robustness of the overall design, and theeffectiveness of the loop stresses and topology in suppressing unwantedresponses.

The second parameter considered for optimization was the length of thestiffening LS strand; tested were lengths of 30, 40, 47, and 55.Fluorescence measurements were made to find the true (false) positiverate from an estimate of the number of hairpins open (closed) when theenzyme is present (absent). The best performing designs have the highesttrue positive rate (TPR or sensitivity) and the lowest false positiverate (FPR or one minus the specificity). All of the tested stiffeningstrands give good performance with the LS30 and LS47 designs being best,with the former excelling in specificity and in maintaining a low FPRover long periods of time, while the latter is preferred for sensitivityand for the fastest response within the resolution of the experiment,which was carried out for periods ranging from 2 to 21 hours. Thegeneral behavior is a rapid rise to a peak followed by a slowdegradation in performance, with again LS47 being best at early timesbut LS30 performing better over longer times because of its relativeimmunity to false positives. Finally, control experiments were carriedout in which the LS stand was either missing entirely (with a ligatedloop) or where it was such that the dsDNA at the nick (in an unligatedloop) could bend easily. In both cases strong false positives wereobserved, with the hairpin being opened by the hybridizing domain evenwhen no endonuclease was present. This shows that the stiffness of theloop is essential to the proper functioning of the transducer.

For loop transducers that respond to endonucleases, another variable tobe considered for optimization is the length of the LC strand thatcomprises the cleavage domain. The specific designs examined had {LC} of10, 20, and 40, with the hairpin labeled with a donor (Cy3) and anacceptor (Cy5) dye. It was found that the system functioned quite wellwith {LC}=20, but shows high false positive rates when {LC}=10 and highfalse negative rates when {LC}=40.

Based on the foregoing, an “optimal” design with {LL}=80, {LS}=30,{LC}=20, {h₁}=8, {h₂}=8, and {□}=15 was investigated. Tests were madenot just with the endonuclease of interest but also with a differentendonuclease (NcoI-HF, targeting CCATGG instead of HaeIII's target GGCC,designed into the transducer) to look for unwanted non-specific signals.FIG. 3A presents results of polyacrylamide gel electrophoresis analysis.Size standards appear in lanes 1 and 8. It was found that in the absenceof the target enzyme (HaeIII) and the output gate, the loop transducerremained intact (lane 2), while in the presence of the target enzyme itcleaved, and the associated increase in electrophoretic mobility wasobserved in lane 3. The analogous experiment with a non-target enzymeNcoI-HF showed no similar transformation as observed in lane 4 (whichwas essentially unchanged from the no-enzyme case of lane 2). Nextintroducing the output gate, there was no change if the target enzymewas not present (lane 5) or a non-target enzyme was present (lane 7),but that the target enzyme instead cleaved the loop transducer (lane 6)with some of the product as observed in lane 3 while the larger fractionformed a slower moving complex resulting from the cleaved looptransducer opening and hybridizing with the hairpin (intense band inlane 5). This was all as desired.

The molecular beacon was used as the output gate to follow the actionspectroscopically with the data presented in FIG. 3B. This assay wasalso attractive in allowing tracking of the temporal evolution (FIG.3C). With both the loop transducer and the output gate present, nochange was seen in fluorescence if either the target enzyme was notpresent (upright triangles) or a non-target enzyme (NcoI-HF) was present(inverted triangles); this is as expected since under these conditionsthe loop transducer would not be cleaved and thus could not activate themolecular beacon. By contrast, in the presence of the target enzyme, theloop transducer would be cleaved, the beacon would be activated, and asharp increase in fluorescence emission was indeed seen (circles) withthe reaction completing in about 40 minutes. This is near-idealtransducer behavior.

As a test of the modularity of the design, a loop transducer identicalto that tested in FIGS. 3A-3C was prepared except that the recognitionsequence was replaced with the target of NcoI-HF (CCATGG). Assessing itsperformance using a gel and fluorescence as above, it was found tofunction quite well, responding strongly to the presence of the targetenzyme but not at all to a non-target enzyme (HaeIII). This demonstratesthe modularity of the design and suggests that the approach could havebroad applicability

Protease Sensing

Proteases are enzymes that cleave peptides, and therefore to create aloop sensor for proteases a peptide segment is preferably be used as thecleavage domain. For example, the protease trypsin targets aneight-residue peptide which was incorporated into the DNA loop. Beforeconducting physical experiments, a molecular dynamics simulation wasperformed in order to look for any undesired behavior, and especiallyfor any unwanted interactions between the peptide and the DNA. Thesimulation confirmed (over its 200 ns duration) that the uncleaved looptransducer stayed in a stressed ‘bow’ configuration as depicted in FIG.1, though of course there were thermal fluctuations. Moreover, thepeptide showed no significant interaction with the DNA, and the peptideexisted in a stretched-out conformation that should be available forattack by the protease.

The system behavior in the real world was examined using both a gel andfluorescence with the results shown in FIGS. 4A (lanes 1-3) and 4B. Thedata shows that the transducer does indeed operate as desired with thehairpin opened only when trypsin is present (lane 2) and not when it iseither not present (lane 1) or when a non-target protease, namely thetobacco etch virus TEV protease, is present (lane 3). In addition, thetransient spectroscopy in FIG. 4B shows more quantitatively thatnear-ideal sensor response is realized, with little leakage when notrypsin is present and with the full response to trypsin occurring inabout 25 minutes. This modularity of the basic sensor system design wasenabled by the non-specific mechanism of the loop stresses and topologythat suppress leakage.

As a second example of a protease transducer, a loop transducer wasproduced that was identical to that described immediately above exceptfor having a cleavage domain for TEV protease (with target sequence ofseven peptides) rather than for trypsin. The data for this transducer isshown in FIGS. 4A (lanes 4-6) and 4C. The gel showed the desiredbehavior when no protease was present (lane 4) and after TEV proteasewas added (lane 6). However, when a non-target protease (trypsin) wasadded, two intense bands were seen (lane 5) indicating that there issignificant interaction of the loop transducer and the output gate. Thisis also seen in the transient fluorescence (FIG. 6C), though there isclearly a much stronger response to TEV protease. It is possible thatthis non-specific behavior of the TEV protease originates not in theloop transducer but in the protease's star activity at the non-optimaltemperature of the experiment.

Logic

As shown herein, one can change target specificity of a system bymodifying the cleavage domain to convert the activity of various enzymesinto signals. An important benefit of performing this conversion is thatthe products of such sensors are readily combined to perform logicaloperations. This makes possible information processing of multiple datastreams, to improve the reliability of the sensor output. Among otherthings, such designs can in principle greatly reduce false positiverates, e.g., by introducing an AND function that requires the presenceof two different nucleases in order to giving a positive response.

One can create a Boolean OR gate or a NAND gate, specifically bycreating two (or more) loop transducers with cleavage domainsappropriate to two (or more) target enzymes but sharing the samesequence in their hybridizing domains that matches a particularmolecular beacon. This idea is illustrated in FIGS. 5A and 5B for a2-input OR-gate that detects either or both HaeIII and NcoI-HF. Uponsensing either or both enzymes, the corresponding loop transducer(s)hybridized to the molecular beacon, resulting in an increase influorescence as seen in FIG. 5C. In this design, the output gate(labeled hairpin) was functionalized with a donor-acceptor dye pair (Cyeand Cy5), and opening of the hairpin resulted in a rise in the donor(Cy3) emission. A NAND functionality is achieved simply by monitoringthe Cy5 dye instead. FIG. 5D gives the emission of the Cy3 dye beforeand 2 hours after the addition of either or both enzymes, and a clearincrease in emission is evident when either or both enzymes are presentin the solution. Interestingly, FIGS. 7C and 7D also show that the DNA“circuit” acts not only as a binary OR gate, but as an analog adder aswell. This same type of approach can be applied to a protease system.

One could feed the DNA output signals from multiple loop transducersinto DNA logic circuits, and thereby achieve a modularity in which thetransduction and logic functions are separated. As illustration, we hereconsider realizing an AND function on the outputs of two looptransducers using the design shown in FIG. 6. When both DNA outputs fromthe loop transducers are present, the labeled reporter duplex wouldself-assemble back into a stabilized hairpin and in that state provide afluorescent output. Gel electrophoresis data showed sufficient resultsof the AND-gate construction.

Further generalizations are possible using methods like those in [4] andin other papers on DNA logic [8].

Amplification

Another use to which one can put the DNA output of the loop transduceris amplification. The process takes advantage of the catalyticcapabilities of DNA oligonucleotides as exploited for example in [5]. Adepiction of what should readily be possible is shown in FIG. 7.

Signal amplification is commonly used with sensors, typically byintroducing gain into the electrical readout circuitry. For sensitivedetection it is best to do this amplification as early as possiblefollowing transduction so as to minimize the amplification of noise.This suggests that for the loop transducers it would best to do theamplification at the molecular level rather than in subsequent opticalor electrical stages. Two types of amplification at the molecular levelcan be considered, with one being inherent in the enzyme itself due toits turnover. The other is DNA amplification either usingenzyme-dependent methods such as the polymerase chain reaction orrolling circle amplification (RCA), or with a DNA-based scheme that isenzyme-free.

The output DNA signals from the loop converter system can benefit fromthe integration of non-enzyme dependent amplification schemes. Due tothe high amplification range of the catalytic hairpin assembly (CHA),the output DNA signals can be programmed as an input/catalyst to triggerthe amplification process of CHA systems. A modified CHA amplificationscheme for the proposed system was tested in the absence of theendonuclease-to-DNA signal converter. Fluorescence results indicate thatthe input signal was amplified at least 4× times using the modified CHAscheme. Since the modified CHA scheme only leveraged theinternal-toehold mediate strand displacement in order to be compatiblewith the existing endonuclease-to-DNA signal converter, it was expectedthat the amplification factor was less optimal compared to those systemsusing the external-toehold mediated strand displacement. To improve theamplification factor, the hairpin structures of the modified CHA schemewere evaluated next. While maintaining the same structure of the firsthairpin, the second hairpin's stem was padded with non-trivial bases to(i) increase its stability and (ii) minimize leaks in the absence of thecatalyst strand. Fluorescence results indicate that the amplificationfactor linearly improved as a function of additional non-trivial bases.It is relevant that the proposed enzyme-to-DNA signal converter can beequipped with an amplification circuit to boost the output signal forlow concentration detection applications.

Nucleic Acid Detection

Rather than as a detector of enzymes, the transducer can alternativelybe used to detect ssDNA or RNA. The idea is that the oligo to bedetected would hybridize to the cleavage domain of a nucleasetransducer, thus making the latter susceptible to attack by anendonuclease and thereby revealing the presence of the target oligo. Toillustrate, we considered an RNA biomarker as the target, with itsbinding to a loop transducer. In theory, any microRNA biomarkers couldbe targeted in this way. Gel electrophoresis and fluorescencespectroscopy confirmed the functionality of this design, with theRNA-DNA hybrid indeed being cleaved by the HaeIII endonuclease, therebyopening the loop and activating the molecular beacon. It is expectedthat a wide variety of nucleic acids could be detected in this way.

Further Embodiments

Beyond the above-demonstrated enzyme loop sensor effective for bothendonucleases and proteases, it should be possible to develop similarschemes for other technologies such as microRNAs and engineered proteinssuch as zinc-finger nucleases.

The use of a peptide nucleic acid (PNA)-based approach should bepossible either as the LS strand for increased rigidity and thermalstability, or as a facile and lower cost means of inserting a peptideinto a DNA loop.

Beyond the demonstrated fluorescence outputs, color-change readoutsshould also be possible, e.g., with the DNA release driving across-linking reaction between particles (e.g., gold or magnesium). Oranother embodiment could involve tethering to metallic surfaces so as togenerate electrical outputs via standard electrochemical methods.

Also contemplated are designs with the hybridized LS and/or LC strandsdirectly attached to the LL loop strand so that they would not get lostand hence could be reconstituted from a dried state.

The proposed system can be tethered to 2D substrates such as lipidbilayers via the cholesterol-labeled DNA oligomer for enhancing speed aswell as utilizing the localization effect for sensing surface-boundbiomarkers.

Signals from this system should be measurable by, circular dichroism(CD), UV-VIS, and excitonic-coupling phenomenon, in addition to thetechnqiues described in the examples.

Quenchers can be fluorescent dyes or other suitable quenchers offluorescence as known in the art.

Advantages

Described herein is a new technique for enzyme detection/transduction byconverting specific enzymatic activity into a DNA signal that can inturn be combined, processed, and/or amplified using known DNA methods.The advantages and new features of the method over existing approachesmay be summarized as follows.

It provides a general technique that can be applied to endonucleases andproteases, and potentially also to many other classes of enzymes. Thisis made possible by the non-specificity of the principle of operation(based on the loop stiffness and topology) and by the demonstratedmodularity of the design. These considerations should make the approachbroadly applicability to many different areas in biomedicine, homelandsecurity, etc.

The simplicity of the loop transducer design makes it scalable and makespossible the processing of other information beyond biomaterials.

With the loop transducer's simple DNA oligo output the technique can bereadily coupled to the world of DNA nanotechnology, and especially tostrand displacement networks. As illustrated in this disclosure, twoprimary functions achievable in this way are logical processing of theoutputs and amplification of the outputs.

The nano-size, non-toxic nature, and robustness to enzymatic attackshould make the approach adaptable to in vivo applications, unlike manyalternatives.

The approach is accomplished at very low cost in view of the relativeease of obtaining the synthesized oligomers commercially. In standardstorage conditions, shelf-life should be excellent given the knownrobustness of DNA.

The proposed system can withstand exonuclease digestion if using thecircular loop for in vivo application.

Concluding Remarks

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

REFERENCES

-   [1] N. Seeman, Structural DNA Nanotechnology (Cambridge Univ. Press,    2016).-   [2] S. Tyagi and F. R. Kramer, “Molecular beacons: Probes that    fluoresce upon hybridization,” Nature Biotech. 14, 303 (1996).-   [3] B. Yurke, “A DNA-fueled molecular machine made of DNA,” Nature    406, 605 (2000).-   [4] G. Seelig, D. Soloveichik, D. Y. Zhang, and E. Winfree,    “Enzyme-free nucleic acid logic circuits,” Science 314, 1585 (2006).-   [5] C. Wu et al., “A nonenzymatic hairpin DNA cascade reaction    provides high signal gain of mRNA imaging inside live cells,” J. Am.    Chem. Soc. 137, 4900-4903 (2015).-   [6] E. Protozanova, P. Yakovchuk, and M. D. Frank-Kamenetskii,    “Stacked-unstacked equilibrium at the nick site of DNA,” J. Mol.    Biol. 342, 775 (2004).

What is claimed is:
 1. A method of detecting enzyme activity, the methodcomprising: providing a sensor system comprising (a) a loop transducercomprising a stiffening domain of about 30 to 55 base pairs in length, acleavage domain cleavable by an enzyme of interest, and a firsthybridizing domain of about 12 to 27 base pairs in length; and (b) anoutput gate comprising a second hybridizing domain of about 8 to 15 basepairs in length and complementary to the first hybridizing domain, afluorophore, and a quencher, wherein the system is configured so that inthe absence of the first hybridizing domain, the quencher quenches thefluorophore, and upon hybridization of the two domains, the quencherbecome separated from the fluorophore sufficiently to allow fluorescencethereof; contacting the sensor system with a sample; allowing the sampleto react with the sensor system; and then measuring fluoresce from thefluorophore, wherein the fluorescence indicates activity of the enzymeof interest.
 2. The sensor system of claim 1, wherein the cleavagedomain is cleavable by an endonuclease.
 3. The sensor system of claim 1,wherein the cleavage domain is cleavable by an protease.
 4. The sensorsystem of claim 1, further comprising a second output gate comprising asecond fluorophore configured as a Förster resonance energy transferpartner of the other fluorophore.